Collection efficiency of mononuclear cells in offline extracorporeal photopheresis: can processing time be shortened?

BACKGROUND: Extracorporeal photopheresis (ECP) is a well-established but lengthy and burdensome cell-based therapy for various diseases such as cutaneous T-cell lymphoma, graft-versus-host disease and organ rejection after transplantation. The number of mononuclear cells (MNCs) that needs to be collected to obtain a clinical response to ECP is still under debate. The purpose of this retrospective study was to determine the number of lymphocytes, monocytes and neutrophils in mononuclear cell products (MCP) by flow cytometry and the collection efficiency in the offline ECP setting. MATERIALS AND METHODS: We collected data from 10 different patients undergoing 162 ECP procedures using the Spectra Optia device for MNC collection. White blood cell (WBC) count of MCP was determined using a hematology analyzer. MNCs were analyzed for CD45 and CD14 expression by flow cytometry to exactly determine the collected lymphocyte and monocyte fractions. RESULTS: Collected MCP showed high cell yields with 55.3×106/kg MNCs and 41.1×106/kg lymphocytes. MCP were characterized by high MNC (81.3%) and low neutrophils (18.7%) percentage. Mean collection efficiency for WBCs and for MNCs was 23.9% and 62.0%, respectively. The MNC fraction showed a moderate to high correlation between peripheral blood cell count of patients and MCP count. DISCUSSION: This study is one of a few reports showing the monocyte-to-lymphocyte relation in MCP for ECP determined by flow cytometry. In comparison to historical data from inline ECP, the offline ECP processing one total blood volume results in considerably higher cell yields. For this reason, and to reduce the burden on patients, we propose that the offline ECP processing time can be substantially reduced.

Epstein-Barr virus reactivation is not causative for post-COVID-19-syndrome in individuals with asymptomatic or mild SARS-CoV-2 disease course.

PURPOSE: Post-COVID-19-Syndrome (PCS) frequently occurs after an infection with severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). However, the understanding of causative mechanisms is still limited. Aim of this study was to determine the PCS rate among SARS-CoV-2 seropositive blood donors as representatives of supposedly healthy adults, who had experienced an asymptomatic or mild COVID-19 disease course, and to examine whether Epstein-Barr virus (EBV) is reactivated in individuals reporting PCS. METHODS: The PCS rate was determined using questionnaires that included questions about infection and persistent symptoms. Pre-pandemic blood samples and samples collected at regular, pre-defined times after a SARS-CoV-2 infection were analysed for neopterin, a marker for antiviral immune responses, by an enzyme-linked immunosorbent assay (ELISA). Additionally, we determined the rate of SARS-CoV-2 anti-N total antibodies using an electrochemiluminescence immunoassay (ECLIA). Furthermore, quantitative real-time polymerase chain reaction (qPCR) to detect EBV DNA and ECLIA screening for EBV viral capsid-antigen (VCA) IgM, IgG and EBV nuclear antigen 1 (EBNA) IgG were performed. RESULTS: Our data reveal that 18% of all infections result in PCS, with symptoms lasting for up to one year. In individuals reporting PCS, no elevated levels of neopterin were detected, indicating no persisting pro-inflammatory, antiviral immune response. SARS-CoV-2 antibody levels were declining in all participants in comparable manner over time, pointing to a successful virus clearance. In individuals with PCS, no EBV DNA could be detected. Furthermore, no differences in EBV specific antibody levels could be shown in PCS groups compared to non-PCS groups. CONCLUSION: Our data suggest that PCS in per se healthy, immunocompetent adults cannot be ascribed to a reactivation of EBV.

SARS-CoV-2 IgG Levels Allow Predicting the Optimal Time Span of Convalescent Plasma Donor Suitability.

Convalescent plasma (CP) has been in use for the treatment of numerous infectious diseases for more than a century, recently also for coronavirus disease 2019 (COVID-19). A major challenge for this treatment is identifying suitable donors with sufficient levels of functional antibodies and to determine the optimal time span for CP donation. In this retrospective study, we analyzed 189 CP donations of 66 donors regarding anti-SARS-CoV-2 anti-S IgG antibody levels. We found a significant correlation between the semi-quantitative SARS-CoV-2 IgG ratio values and in vitro antibody functionality. A time-to-event analysis allowed us to predict the optimal time span of COVID-19 CP donor suitability. We found that high IgG ratio values, which significantly correlate with high in vitro antibody functionality, were suitable for CP donation for a median of 134 days after the first CP donation. Donors with lower IgG ratios were suitable for a median of 53 days. Our data support plasma collection centers to determine optimal points in time for CP donation by means of widely used semi-quantitative laboratory IgG ratio values.

Persistence of Naturally Acquired and Functional SARS-CoV-2 Antibodies in Blood Donors One Year after Infection.

The developmental course of antibodies produced after a SARS-CoV-2 infection has been insufficiently investigated so far. Therefore, the aim of this study was to investigate the dynamics of SARS-CoV-2 antibody levels against the viral nucleocapsid- and spike-protein among Austrian blood donors as a representative group of a supposedly healthy population within the first year after a SARS-CoV-2 infection. The impact of age, sex, vaccination status, AB0-blood group and awareness about the infection was evaluated. Our study shows that the level of anti-N antibodies is declining, while anti-S antibody levels remain stable. Antibodies detected were functional in vitro. Age, sex and blood group do not influence antibody dynamics. However, blood group AB shows significantly lower antibody levels and in vitro functionality compared to other blood groups. Our data reveal that one out of five individuals was not aware of a previous SARS-CoV-2 infection and that the disease course neither affects the level of antibody production nor the in vitro functionality. We also found that 14% of participants show persisting COVID-19-related symptoms for up to nine months. Our results provide valuable insights into the dynamics of the immune response after a SARS-CoV-2 infection in a representative cohort of adult blood donors in Central Europe.

Jknull alleles in two patients with anti-Jk3.

BACKGROUND: As of publication, a total of 41 null alleles have been acknowledged by the International Society of Blood Transfusion (ISBT) to cause the rare Jknull phenotype, but none have been discovered in Austria thus far. MATERIALS AND METHODS: Two patients with anti-Jk3 were serologically identified by a positive antibody screening and typed as Jk(a-b-). The initial genotyping using an SSP-PCR method for the common 838A/G polymorphism indicated a JK*02/02, or JK*01/02 genotype, respectively. To find the disruptive mutations, Sanger sequencing was performed and results were compared to the reference sequence. The patient's antibodies were characterized with a monocyte monolayer assay (MMA) for their potential clinical significance. RESULTS: Three novel null-mutations of the SLC14A1 gene were found in two patients. Patient 1 was homozygous for a 10bp deletion in exon 4 (c.157_166del on JK*02). Testing of her family members revealed Mendelian inheritance of the deletional allele. The other patient was compound heterozygous for two mutations: one allele carrying a single base deletion in exon 4 (c.267delC on JK*01) and the other a splice site mutation in intron 3 (c.152-1g>a on JK*02). The MMA results suggest high clinical significance of the anti-Jk3 in both patients. DISCUSSION: The detected mutations led to Jknull phenotypes and are the first description of JKnull alleles in the Austrian population.

Single-use IgE-selective immunoadsorber column for the treatment of severe atopic dermatitis.

BACKGROUND: Atopic dermatitis (AD) is a chronic and relapsing inflammatory skin disease with unmet therapeutic need in a critical cohort of recalcitrant cases. Immunoadsorption (IA) aims at an immunomodulatory depletion of pathogenic serum mediators and has recently revealed promising clinical results for the treatment of AD. OBJECTIVE: To determine efficacy, sustainability, safety, and clinical impact of IgE selective IA in AD using a single-use IgE immunoadsorber column. METHODS: This open-label pilot study comprised five patients (mean SCORAD 67.9 ± 11.4, range 52.2-81.9; mean serum IgE level 5904 ± 5945 U/mL, range 1000-15 600 IU/mL) who underwent IgE-selective IA. Three patients continued prior therapy with systemic immunosuppressive drugs during IA as an add-on therapeutic approach. All patients received three courses of IA. The first course consisted of three consecutive daily treatments followed by two sequences with two consecutive applications. All courses were performed on a monthly regimen. RESULTS: IA proved efficacy in selectively depleting serum IgE levels in all participants (mean reduction by cycle of 81% ± 12%, range 64%-93%). It further led to a clinically relevant and sustained improvement of AD with a maximum decline in SCORAD and EASI scores by up to 35% and 52%, respectively, compared to baseline. Scores persisted below baseline for at least 12 weeks beyond the last IA. The intervention was also well tolerated with no severe adverse events during a total of 35 procedures. CONCLUSION: Data of this preliminary trial indicates clinical efficacy, feasibility, safety as well as tolerability of IgE-selective IA in AD.